Summary/Abstract The goal of this proposal is to elucidate a regulatory role for type-I interferon (IFN) at the blood brain barrier (BBB) during West Nile virus (WNV) encephalitis. WNV is a mosquito-borne flavivirus, now endemic in North America. Over the past decade, WNV has caused a growing epidemic of lethal viral encephalitis in the United States. West Nile pathogenesis, particularly how the pathogen infiltrates the central nervous system (CNS), remains poorly understood. Under normal conditions, the CNS is protected from pathogens in the circulation by the BBB, an interface consisting of brain microvascular endothelial cells (BMEC's) joined by tight junctions and supported by adjacent astrocyte endfeet [1]. However, WNV is able to cross the BBB through unknown mechanisms, and establishes infection in neurons and other parenchymal CNS tissues [2, 3]. CNS infection results in immune activation which dysregulates the BBB, facilitating the access of essential antiviral leukocytes to the CNS during infection; however, trafficking of infected leukocytes to the CNS may also provide an opportunity for neuroinvasion by WNV [4, 5]. Moreover, while trans-BBB immune trafficking is necessary for CNS viral clearance, the vulnerability of the CNS to damage and its limited capacity for repair can result in significant bystander injury and immunopathology during antiviral immune responses. Thus, the BBB must be tightly regulated during CNS infection, with a balance between facilitating access of antiviral leukocytes to sites of infection and protecting the CNS from immune-mediated damage and potential pathogen exposure from circulating virus and infected host immune cells. Antiviral cytokine signaling is a likely mechanism by which this balance is achieved. As a major component of the systemic immune response to WNV, the innate immune cytokines of the type-I IFN family are of critical importance in protecting both CNS and peripheral tissues from infection, via both direct antiviral activity and promotion of adaptive immune responses [6-8]. Interestingly, type-I IFN's have additionally been shown to exert anti-inflammatory properties at the BBB in the context of CNS autoimmunity, strengthening endothelial barrier integrity and impeding the ability of leukoyctes to bind endothelium and migrate into the CNS [9, 10]. However, the actions of type-I IFN at the BBB during viral encephalitis have not yet been investigated. Preliminary studies in our laboratory using in vitro models of the BBB have shown that the induction of type-I IFN signaling in both BMEC's and astrocytes after WNV infection results in enhanced endothelial barrier function and decreased transendothelial viral and immune trafficking. Building on these in vitro findings, we propose to use an established murine model of WNV encephalitis in mice with cell-specific deletions of the type-I IFN receptor (IFNAR) in endothelial cells or astrocytes in order to better understand how type-I IFN signaling at the BBB impacts neuroinvasion and CNS viral pathogenesis. In addition, we will examine potential regulatory effects of type-I IFN on CNS antiviral immune trafficking and consequent viral clearance and/or immunopathology. We hypothesize that type-I IFN is a key regulator of the BBB during WNV infection, preserving barrier integrity, restricting access of WNV to the CNS, and limiting CNS immune infiltration and damage during WNV encephalitis.